This proposal is an extension of ongoing research designed to provide basic information on the structure and function of the Mycobacterium tuberculosis. One goal is to understand how the insertion sequence is integrated into the genome of the M. tuberculosis complex and to evaluate its mobility. Previously identified repeats will be characterized and other insertion sequences will be isolated. Insertion sequences may be important in promoting,rearrangements in the chromosome and in altering gene expression. Regardless of their function, repeated sequences provide insight into the evolutionary relationship of these organisms. Another goal is to examine-genetic differences between M. tuberculosis and M. bovis to understand the significance of IS6110 copy number. A third goal is to isolate putative virulence genes from M. tuberculosis H37Ra. The specific aims are: 1) to demonstrate that IS6110 functions as a mobile element by showing that it can transpose, and to determine the frequency with which such insertions occur. 2) To determine if the sites of IS6110 insertion are conserved within individual strains, species, and across species line and if the IS6110 insertion is target site specific. This will involve isolating DNA flanking IS6110, using these flanking sequences to probe genomic blots, and DNA sequencing of the flanking regions. 3) To characterize two previously identified repeated sequences and several of the insertion elements. 4) To analyze genetic differences between M. tuberculosis and M. bovis by fingerprinting the rRNA genes and DNA gyrase B genes to determine the basis for the difference in copy number of IS6110 in these organisms, and to compare phenotypic characteristics with copy number. 5) To determine whether the IS6110 insertions in M. tuberculosis H37Ra correlated with the loss of virulence. This will include searching for the uninterrupted genes in M. tuberculosis H37Rv, transforming H37Ra with these putative virulence genes, demonstrating virulence in mice, and identifying gene products.